This project will employ homology modeling and protein analysis algorithms developed at LLNL (the Protein Pipeline) to predict and analyze the structures of each botulinum neurotoxin (BoNT) subtype using the sequences of the proteins determined in Project 1. Sequence-structure alignments will be generated using a variety of algorithms, and the backbone of the model will be created using our AS2TS modeling package. The known crystal structures of the BoNT/A and BoNT/B holotoxins and domains will be used as templates. The construction of the terminal regions and loops, ammo acid insertions and deletions, and template-model structure comparisons will be performed using the LGA program. The collection of predicted subtype structures will be compared with the known toxin crystal structures to identify those subtypes that are most likely to exhibit functional differences, and this information will be used to identify which BoNT variants should be crystallized and carried forward for structure determination in Project 3. Each subtype toxin protein sequence will also be blasted against a database containing all known protein sequences using our Protein Pipeline to identify those peptide sequences (signatures) that are not found in any other protein and are unique to the subtype. The signatures and mutations will be mapped onto the predicted structures and examined to identify those subtype-specific peptide signatures that are sufficiently accessible to be used as epitopes for the development of BoNT subtype-specific antibodies by other key collaborators.